Study On Co-transformation Of CsLEA2/CsALDH12A1 And Bar Genes Into Medicago Sativa L.

Abiotic stress, especially drought and saline stress are two major factors that limiting the growth of. Medicago sativa L. Using the transgenic technology to develop superior alfalfa new lines with improved stress and herbicide tolerance has the vital practical significance. In our previous study CsLEA2 and CsALDH12Al have been cloned from Cleistogenes songorica, a xerophytic desert turfgrass, and were stress induced expression in Arabidopsis thaliana which enhanced drought tolerance. In this study, bar and CsLEA2/CsALDH12A1 were co-transformed into legume alfalfa and the drought and salt tolerance of TO generation of transgenic alfalfa were tested. The main results are as following: 1. pEarlygate101 Bar-35S::CsLEA2 and pEarlygate101 Bar-35S::CsALDH12A1 were transformed into Medicago sativa cv Jinhuanghou by Agrobacterium-mediated method. In total, we collected 119 transgenic alfalfa with CsLEA2 gene and 90 transgenic alfalfa with CsALDH12A1 gene.2. Transgenic plants were preliminary screened by spraying 1mL/L 10% Basta solution,40 transgenic alfalfa with CsLEA2 gene and 54 transgenic alfalfa with CsALDH12A1 gene resistant to herbicide. All these transgenic lines were conducted further molecular authentication through PCR and RT-PCR. There are 39 resistant plants by integrating CsLEA2 gene and 16 resistant plants by integrating CsALDH12A1 gene, which can be amplified with targeted fragment, and all of them could express effectively in transgenic alfalfa. PCR positive rate is 97.5% and 29.6%, respectively.3. TO generation of transgenic alfalfa plants were treated with drought and salt stress. The study of expression patterns of CsLEA2 and CsALDH12A1 during drought and salt stress showed that the drought and salt stress induced higher expression in leaf. Compared with control, the CsLEA2 expression level in leaf at 15 d drought stress and 10 d of 200 mM NaCl treatment are 5.57 times and 4.66 times, CsALDH12Al expression level are 6.11 times and 6.87 times, respectively.The phenotype observation indicated that the leaves of wild-type alfalfa turned yellow after 15 d of drought stress, while transgenic plants showed wilt and resume growth after rewatering. Transgenic plants also showed better growth than the control after 10 d of salt stress. These suggested that overexpression of CsLEA2 and CsALDH12A1 gene in alfalfa showed a better drought and salt tolerance.Determination of physiologic and biochemical indices under drought and salt stress showed that the biomass, the leaf water content, chlorophyll content, chlorophyll fluorescence Fv/Fm, Pn and proline content of wild-type alfalfa are lower than the transgenic alfalfa. However the MDA content was significantly higher than transgenic alfalfa (P<0.05). In addition, overexpression of CsLEA2 and CsALDH12A1 gene showed a relative lower Na+ content in leaves of transgenic plants to reduce ion toxic effects.